1. Field of the Invention
The present invention relates to lipid A analogs characterized by the replacement of a sugar unit by a derivative of pentaerythritol. It also relates to analogs of carbohydrate ligands, including lipid A, characterized by the replacement of an amino sugar unit by a derivative of pentaerythritylamine.
2. Cross-Reference to Related Applications
Biomira (Jiang, et al.), PCT/US00/31281, filed Nov. 15, 2000 relates to the design and synthesis of some new Lipid-A analogs. The analogs were monophosphorylated, and contained either (1) at least one novel and unnatural lipid (such as lipids I or II) of compounds 33 and 102 (its FIG. 3), or (2) an unnatural combination of lipids. The latter refers to those Lipid-A analogs that carry lipids of uniform chain length. Its Compounds 54 and 86 (its FIG. 4) fall into this category. Its Compound 70 (its FIG. 19) is similar, but it also contains an n-propyl group at 3-O-position and is an example of Lipid-A analog that incorporates a short unnatural alkyl group with an unnatural ether linkage. By using a synthetic lipopeptide antigen, (FIG. 34), a modified 25-amino-acid sequence that is derived from tumor-associated MUC1 mucin, the applicants were able to evaluate the adjuvant properties of certain synthetic Lipid-A analogs disclosed in this invention. Based on the data of T-cell blastogenesis and IFN-γ level obtained through preliminary in vivo/in vitro studies, it was demonstrated that synthetic Lipid-A structures 48, 54, 70, 86, 102 and 104 are as effective, as adjuvants, as the Lipid-A preparations of bacterial origin.
Koganty, et al., U.S. Prov. Appl. No. 60/387,437, filed Jun. 11, 2002 relates to combinatorial peptide and glycopeptide libraries utilizing a pentaerythritol core.
Biomira (Koganty et al.), PCT/US03/10750, filed Apr. 9, 2003 teaches that a glycolipopeptide comprising at least one disease-associated epitope, and characterized by at least one lipidated interior amino acid or by the presence of a MUC1 epitope, may be used in a vaccine, preferably in conjunction with a liposome.
Biomira (Longenecker, et al.), PCT/US95/04540, filed Apr. 12, 1995, discloses that a conjugate of a primary epitope and an immunomodulatory peptide, or a mixture of a primary antigen and an immunomodulatory peptide, may be used to elicit an immune response which is CMI-specific.
Biomira (Jiang et al.), PCT/CA03/00135, filed Feb. 4, 2003, relates to the use of covalently lipidated oligonucleotides comprising the CpG dinucleotide unit, or an analogue thereof, as immunostimulatory agents. It discloses that a Pet structure can be used to link together such units.
The above-noted related applications are hereby incorporated by reference in their entirety.
3. Description of the Background Art
Pentaerythritol. Pentaerythritol (Pet) and di-pentaerythritol (di-Pet) are common polyols and they are widely used in oil industry to produce lubricants and other macromolecules. A derivative, tetrakis-[13-(2′-deoxythymidin-3′-O-yl)-6,9-diaza-2-oxa-5,10,13-trioxotridecyl)-methane (dT4-PE-PLC) has been used as a liquid phase carrier for large-scale oligonucleotide synthesis in solution (Wörl, R. et al, 1999, compound 6). In addition, Pet derivatives, semifluorinated pentaerythritol tetrabenzoates, have been employed to design liquid crystalline structures (Cheng, X. H. et al, 2000) and pentaerythritol lipid derivatives (e.g., dimristoyl-trimethylglycine pentaerythritol) have been used in the preparation of cationic liposomes for the delivery of nucleic acids into mammalian cells (Nantz, M. H. et al, 2001). A triamine derivative of pentaerythritol has been used as a starting material in the preparation of chelating agents (Dunn, et al., 1990).
The four-directional core (the “Pet” unit) of pentaerythritol has been employed successfully as a coupling agent, for example, in the synthesis of multifunctional dendrimers (Armspach, D. et al, 1996 and Kuzdzal, S. A. et al, 1994), and as a molecular scaffold for combinatorial chemistry (Farcy, N. et al, 2001). Furthermore, Ranganathan et al used the Pet unit as a core to design a spiro-self-assembling cyclic peptide for constructing twin nanotubes (Ranganathan, D. et al, 2001).
It is particularly interesting to note the use of the Pet unit to couple sugar units. Lindhorst, et al, Eur. J. Org. Chem., 2027-34 (2000) used the Pet unit as a framework for a cluster of four mannosides. Schmidt, et al., Eur. J. Org. Chem., 669-674 (2002) prepared similar structures in which a lipid group (C16H33) was O-linked to one of the four peripheral carbons, and one to three mannoside residues were O-linked, through an ethyleneoxy oligomeric spacer, to other of the peripheral carbons. Those peripheral carbons which did not link to a lipid or to a sugar-containing moiety were simply hydroxylated. Finally, Hanessian et al. 1996 used a pentaerythritol scaffold to present a cluster of two Tn (the monosaccharide GalNAc) or TF (the disaccharide D-Galβ(1->3)GalNAc) epitopes, each O-linked through a spacer to a peripheral carbon of the Pet core. Of remaining two peripheral carbons, one was O-linked to —CH2CH2NHAc, and the other O-linked to either allyl (Hanessian 33) or 1-octenyl (Hanessian 37). In none of these references was a peripheral carbon of the Pet core N-linked to any moiety.
In the various applications mentioned above, the Pet unit serves as a core to carry other moieties. It may also be used to replace a sugar unit in an oligosaccharide. However, it has never before been used to replace a sugar unit in the lipid A disaccharide. Nor has Pet-NH— been used to replace an amino sugar in any carbohydrate ligand.
Toepfer et al disclosed sialyl-Lewis X and sialyl-Lewis A mimics containing one Pet unit (Toepfer et al. 1995; Toepfer et al. 2000) as new ligands for selectin binding. Thus, in compound 4 of Toepfer et al. 1995, two of the peripheral carbons of the Pet unit are hydroxylated, one is O-linked to a moiety comprising a single sugar unit, and the last one is O-linked to a moiety comprising a disaccharide. It should be noted that in Toepfer's analogs, the Pet unit replaces a normal sugar unit, not an amino sugar as in applicants' carbohydrate ligand analogs. In addition, the only lipophilic groups contemplated by Toepfer et al. are groups customarily used as protecting groups in organic synthesis, such as those resulting in replacement of sugar hydroxyls with —O-All, —O-Tf, or —O-Bn.
Aguilera et al 1998 reported the testing of analogs of oligosaccharides for anti-mitotic activity. The original oligosacccharides were the tetrasaccharide α-D-GalNac-β-D-Gal-(1->4)-[α-L-Fuc-(1->3)]-β-D-GlcOMe, and a related sulfated trisaccharide (Aguilera compound 1), which contain a Lewis X-type structure. In the analogs of the trisaccharide (Aguilera compounds 13-16), one sugar was replaced with a Pet unit. In the analogs of the tetrasaccharide (17, 18), two of the sugar units were replaced with Pet units. The analogs thus contained the disaccharide in which the β-fucosyl residue was linked to the C-3 position of the GlcNac. In all six analogs, one hydroxyl of the disaccharide moiety was replaced with —O(CH2)7CH3, thus imparting a lipid function. In analogs 14, 16 and 18, three of the four Pet unit peripheral carbons were hydroxylated (the remaining carbon being linked to a group comprising the disaccharide moiety). In Aguilera compounds 13, 15 and 17, two peripheral Pet carbons were hydroxylated and the third was sulfated. However, these compounds were found to be inactive as antimitotic agents in all of the cell types, thus discouraging further use of negatively charged groups in analogs of this family.
Lipopolysaccharide (bacterial). Lipopolysaccharide (LPS) is a unique glycolipid found exclusively in the outer leaflet of the outer membrane of Gram-negative bacteria. Structurally, bacterial LPS molecule has three main regions: the O-antigen region, the core region and the Lipid-A region (Stryer, 1981; Raetz, WO86/05687). The O-antigen region is a strain-specific, polysaccharide moiety and determines the antigenic specificity of the organism. The core region is an oligosaccharide chain and may play a role in maintaining the integrity of the outer membrane. The Lipid-A region is conserved and functions as a hydrophobic anchor holding lipopolysaccharide in place.
LPS is known to trigger many pathophysiological events in mammals, either when it is injected or when is accumulated due to Gram-negative bacterial infection. Before the discovery of Lipid-A component of LPS the term “endotoxin” was generally used to describe the effects of the LPS. The endotoxin from Gram-negative bacteria is heat-stable, cell associated, pyrogenic and potentially lethal. In addition to its endotoxic activities, LPS also exhibits various biological activities, which include immuno adjuvant activity, B-lymphocyte mitogenesis, macrophage activation, interferon production, tumor regression, etc. While both the O-antigen and the core regions modulate the toxic activity of the LPS, it is generally believed that the hydrophobic Lipid-A moiety is responsible for these pathophysiological effects of the endotoxin (Rietschel, 1992: Takada, 1992).
Lipid A and Its Synthetic Analogs. Lipid A is the lipid anchor of lipopolysaccharide (LPS), the outer cell membrane component of Gram-negative bacteria. LPS is a strong activator of the innate immunity of the host following bacterial infection, and its lipid A moiety has been shown to be responsible for the biological activities of LPS in most in vitro and in vivo test systems. The structure-activity relationships of lipid A and its analogs have been extensively studied over the last two decades (Rietschel et al, 1996; Takada & Kotani, 1989).
Lipid-A consists of a β-(1,6)-linked D-glucosamine disaccharide phosphorylated at 1-O- and 4′-O-positions. Hydroxylated and non-hydroxylated fatty acids are linked to the hydroxyl and amino groups of the disaccharide to confer hydrophobicity to the Lipid-A. FIG. 1 of PCT/US00/31281 shows two examples of natural Lipid-A structures, compound A (Imoto, 1985a, b) isolated from E. coli, and compound B (Rietschel, 1984a, b; Seydel, 1981; Strain, 1985) isolated from Salmonella strains.
A large number of synthetic lipid A analogs have been prepared. For example, Lien et al. 2001 describe the agonist ER-112022, in which the disaccharide backbone of lipid A is replaced with —CH2CH2—NHCO—(CH2)4—CONH—CH2CH2—. The two phosphate groups link this substitute backbone to the lipid chains. Christ et al. 1995 prepared the lipid A antagonist E5531, derived by modification of the structure of the endotoxin-antagonistic Rhodobacter capsulatus lipid A, in which the naturally occurring acyl linkages at the C-3 and C-3′ carbons were replaced with ether linkages, and the C-6′ hydroxyl group was blocked. E5531 had advantages in stability and purity.
Takada and Kotani have conducted a thorough study of structural requirements of Lipid-A for endo-toxicity and other biological activities (Takada & Kotani, 1989), comparing synthetic Lipid-A analogs prepared by various groups (Kotani, 1986a, b; Kiso, 1986: Fujishima, 1987; Charon, 1985: Sato, 1995). They reported that for immunoadjuvant activity, the structural requirements of Lipid-A do not appear to be as rigid as those required for endotoxic activity and IFN-α/β or TNF-alpha inducing properties (Takada, 1989; Ribi, 1982). Removal of all fatty acids, however, abrogates all biological activities normally attributed to Lipid-A.
Ribi et al 1982 showed that the minimal structure required for toxicity was a bisphosphorylated β-(1,6)-linked di-glucosamine core to which long chain fatty acids are attached. It appears that an optimal number of lipid chains, in the form of either hydroxy acyl or acyloxyacyl groups, are required on the disaccharide backbone in order to exert strong endotoxic and related biological activities of Lipid-A (Kotani, 1986a).
In addition, removal of either phosphate group results in significant loss of toxicity without a corresponding loss of adjuvant activity. Bioassays on monophosphoryl Lipid-A showed that, while it was 1000 times less potent on a molar basis in eliciting toxic and pyrogenic responses, it was comparable to diphosphoryl Lipid-A (and endotoxin itself) in immunostimulating activities (Werner, 1996). It is known that the diphosphoryl Lipid-A from E. coli and Salmonella strains are highly toxic, but the monophosphoryl Lipid-A from E. coli has reduced toxicity while retaining the numerous biological activities that are normally associated with LPS (Werner, 1996; Takayama, 1984; Ulrich, 1995; Myerr, 1990).
Recently, it was suggested that the agonistic and antagonist activity of lipid A were governed by the intrinsic conformation of lipid A, which in turn was defined mainly by the number of charges, the number and distribution of acyl chains in the molecule (Seydel et al 2000; Schromm et al, 2000).
Furthermore, lipid A has been suggested to be a ligand for Toll-like receptor 4 (TLR4), a pattern-recognition receptor involved in the mediation of immune responses to LPS/lipid A (Kutuzova et al, 2001).
There is a need for effective treatment for Lipid-A/LPS associated disorders, and for a potent adjuvant without the associated toxicity. The high toxicity of unmodified Lipid-A from natural source discourages its general use as a pharmaceutical.
Another major drawback with the naturally derived Lipid-A is in accessing sufficient material with pharmaceutically acceptable purity, reproducible activity and stability. Naturally derived Lipid-A is a mixture of several components of cell wall including those of Lipid-A with varying number of lipid chains. Such heterogeneity in natural Lipid-A product is attributed to two sources: (1) biosynthetic variability in the assembly of the Lipid-A moiety and (2) loss of fatty acids from Lipid-A backbone during processing and purification.
Consequently, it is difficult to control the manufacturing process in terms of reproducibility of composition of the mixture, which has significant bearing in biological activity and toxicity.